Systematic Comparison of the T7-IVT and SMART-Based RNA Preamplification Techniques for DNA Microarray Experiments Jochen Wilhelm, 1* Jai Prakash Muyal, 1 Johannes Best, 1 Grazyna Kwapiszewska, 1 Maria Magdalena Stein, 1 Werner Seeger, 2 Rainer Maria Bohle, 1 and Ludger Fink 1 Background: Small biological samples obtained from biopsies or laser microdissection often do not yield sufficient RNA for successful microarray hybridization; therefore, RNA amplification is performed before mi- croarray experiments. We compared 2 commonly used techniques for RNA amplification. Methods: We compared 2 commercially available meth- ods, Arcturus RiboAmp for in vitro transcription (IVT) and Clontech BD SMART TM for PCR, to preamplify 50 ng of total RNA isolated from mouse livers and kidneys. Amplification factors of 3 sequences were determined by real-time PCR. Differential expression profiles were compared within and between techniques as well as with unamplified samples with 10K 50mer oligomer- spotted microarrays (MWG Biotech). The microarray results were validated on the transcript and protein levels by comparison with public expression databases. Results: Amplification factors for specific sequences were lower after 2 rounds of IVT than after 12 cycles of SMART. Furthermore, IVT showed a clear decrease in amplification with increasing distance of the amplified sequences from the polyA tail, indicating generation of smaller products. In the microarray experiments, repro- ducibility of the duplicates was highest after SMART. In addition, SMART-processed samples showed higher correlation when compared with unamplified samples as well as with expression databases. Conclusions: Whenever 1 round of T7-IVT does not yield sufficient product for microarray hybridization, which is usually the case when <200 ng of total RNA is used as starting material, we suggest the use of SMART PCR for preamplification. © 2006 American Association for Clinical Chemistry Microarray expression profiling experiments usually re- quire RNA in larger quantities than are available from small biological samples (1, 2). Consequently, methods have been developed to amplify the RNA before its use in microarray experiments. The most frequently used method (3) introduces a 5'-terminal promoter (comple- mentary) sequence for the T7 RNA polymerase attached to an oligo(dT) primer into the produced cDNA. After second-strand synthesis, the sense cDNA strand is used as a template for in vitro transcription (IVT). 3 The RNA polymerase produces multiple copies of antisense RNA (aRNA) with amplification factors of up to 1000-fold. Because of random priming to synthesize the second strand, the IVT products are not full-length copies, and the 5'-terminal sequences of the transcripts are underrep- resented in the aRNA. This procedure works well when the initial amount of total RNA is 1–3 g and is used extensively in experiments with cDNA and short-oligo- nucleotide microarrays (4, 5). When less RNA is available, the aRNA yield can be increased by a second round of amplification (6, 7). PCR-based amplification [switching mechanism at the 5' end of the RNA transcript (SMART TM )] was originally developed to amplify full-length cDNAs for construction of clone libraries (8, 9), and more recently has been used in microarray experiments (10 –12 ). cDNA synthesis is performed with an oligo(dT) primer with an attached Departments of 1 Pathology and 2 Internal Medicine, Justus-Liebig-Univer- sity Giessen, Giessen, Germany. * Address correspondence to this author at: Department of Pathology Justus-Liebig-University Giessen, Langhansstrasse 10, 35392 Giessen, Ger- many. Fax 49-641-9941164; e-mail jochen.wilhelm@patho.med.uni-giessen.de. Received October 20, 2005; accepted March 24, 2006. Previously published online at DOI: 10.1373/clinchem.2005.062406 3 Nonstandard abbreviations: IVT, in vitro transcription; aRNA, antisense RNA; SMART, switching mechanism at the 5' end of the RNA transcript; dsDNA, double-stranded DNA; Ct, threshold cycle; Gapd, Mus musculus glyceraldehyde dehydrogenase gene; UTR, untranslated region; and Pbgd, Mus musculus porphobilinogen deaminase gene. Clinical Chemistry 52:6 1161–1167 (2006) Automation and Analytical Techniques 1161